Prolongation of murine hybridoma cell survival in stationary batch culture by Bcl-xL expression

While the ectopic expression of the anti-apoptoticprotein Bcl-2 has been shown to significantly increaseboth cell viability and antibody production in batchculture, some cell lines are refractory to thesemanipulations. For example, the NS/O and theP3x63Ag8.653 murine myelomas, which express highendogenous levels of the Bcl-2 homologue Bcl-xL, areboth resistant to the anti-apoptotic effect of Bcl-2.This indicates that, in these cells, Bcl-2 and Bcl-xLmay be functionally redundant. In order to define therole which Bcl-xL plays in hybridoma cultures, we usedthe Sp2/0-Ag14 cell line. This murine hybridomaexpresses low levels of Bcl-xL and is highly sensitiveto apoptosis induction by cycloheximide (CHX) and byamino acid depletion. Bcl-xL-transfected Sp2/0-Ag14cells were more resistant than the wild type and theplasmid-containing cells to apoptosis induced by CHXand by glutamine depletion. Moreover, when compared tothe vector-transfected control, Bcl-xL-Sp2/0 cellsexhibited a substantial increase in viability instationary batch culture. Interestingly, Sp2/0-Ag14cells overexpressing Bcl-xL showed a growth behaviourthat was similar to the parent myeloma cell lineP3x63Ag8.653. Our results suggest that Bcl-xLexpression levels are sufficient to account for therelative robustness of some hybridoma cell lines instationary batch cultures.     

Protection of hybridoma cells against apoptosis by a loop domain-deficient Bcl-xL protein

The ectopic expression of several members of the Bcl-2 family of anti-apoptotic proteins is a promising strategy to improve the viability of hybridoma cells in culture. However, the impact of post-translational modifications on the function of these proteins in murine hybridomas is unknown. To address this issue, the anti-apoptotic properties of a mutant of Bcl-xL devoid of the so-called “loop domain„ (Bcl-xL▵ 46-83) were investigated using the Sp2/ O-Ag14 hybridoma model. Clones of Sp2/ O-Ag14 cells expressing Bcl-xL▵ 46-83 exhibited resistance against L-glutamine deprivation to similar levels than cells expressing the wild type protein. In contrast, protection against the cytotoxic effects of cycloheximide (CHX) was highly dependent on the level of expression of the Bcl-xL▵ 46-83 mutant. Analysis of the growth behaviour of the transfected cells showed that Bcl-xL▵ 46-83 was superior to the wild type protein in prolonging Sp2/ O-Agl4 cell viability in stationary batch culture. Furthermore, the prolongation of cell viability in batch culture was directly proportional to the level of expression of the mutated protein. Our results indicate that removal of the loop domain improves the anti-apoptotic activity of Bcl-xL in hybridoma cells grown in stationary batch culture. This revised version was published online in August 2006 with corrections to the Cover Date.     

Enhanced formation of mouse hybridomas without hat treatment in a serum-free medium

Summary A newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterinthymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.     

A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines.

We have isolated a subclone of the mouse myeloma cell line P3-X63-Ag8 that does not express immunoglobulin heavy or light chains. This clone X63-Ag8.653 can be used for efficient fusion with antibody-forming cells to obtain hybrid cell lines producing pure monoclonal antibodies. Screening of hybrid cell lines for specificity and immunoglobulin classes was done with a modified enzyme-linked immunosorbent assay.     

Saturable ammonium ion transport in myeloma and hybridoma cells is mediated by the Na+K+2Cl--cotransporter

The total entry of ammonium ions into Sp2/0-Ag14 myeloma cells and hybridoma cells consists of a saturable and a non-saturable component. The plasma membrane Na+K+2Cl--cotransporter was identified as the saturable ammonium ion transporter in both cell lines, and the non-saturable entry was due to simple diffusion of ammonium ions. The theoretical maximum transport rate via the Na+K+2Cl--cotransporter was identical in the two cell lines, but the ammonium ion diffusion rate was considerably higher in the hybridoma cells. We speculate that this is an effect of different membrane properties caused by dissimilar expression of tumour characteristics.     

Expression of a 72-kDa major allergen of American cockroach in hybridoma cell Sp2/0-Ag14

p72 is one of the major allergens of American cockroach. The gene of p72 was cloned into pg2bVH, replacing the VH region, to yield pg2b-p72 which was then transfected into hybridoma cell Sp2/0-Ag14. Western blot analysis of the culture supernatant from Sp2/0-Ag14(p72) detected a diffused protein band (ca. 72 kDa) which appeared to contain heterogeneous glycoforms.     

Extension of Sp2/0 hybridoma cell viability through interleukin-6 supplementation

Sp2/0 hybridoma cells die principally by apoptosis in batch culture. We have found that cultures of the Sp2/0 hybridoma exhibit increased viability in response to interleukin 6 (IL-6) supplementation relative to control cultures during serum shiftdown experiments. When shifted from a medium containing 10% fetal bovine serum (FBS) to a medium with 1% FBS, IL-6 supplemented cultures displayed viabilities and viable cell densities similar to control cultures containing 10% FBS. The degree of the survival response induced varied in accordance with the severity of the shiftdown, as cells resuspended in a high serum medium showed little observable enhancement in viability. The extension in culture viability was not accompanied by an observable decrease in growth relative to control cultures, indicating that the effect was not a consequence of growth inhibition. These results suggest the existence of serum components with behavior functionally similar to IL-6, with respect to enhancing cell survival, and that under certain experimental conditions IL-6 serves as a survival factor. In contrast to the extended viability displayed by cultures supplemented with IL-6, Sp2/0 cultures transfected with IL-6 cDNA expression vectors displayed a growth inhibitory response relative to control cultures. This inhibitory response was characterized by an extended lag phase following inoculation, and a decrease in batch culture cell yield. The depression in cell yield varied with serum concentration, with the largest depression occurring at high serum concentrations. We conclude that interactions between components in serum, presumably growth factors, and cytokines play an important role in altering the behavior of industrially relevant cell lines in culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 439-446, 1997.     

Demonstration of non-histone nuclear proteins in mouse myeloma. Trial comparative study

The non-histone chromosomal proteins from mouse myeloma cell line X 63-Ag 8.653 have been studied using two dimensional polyacrylamide gel electrophoresis. Our results were compared to previous analysis of other myeloma cell lines.     

Production of monoclonal antibodies against the purified glycosylphosphatidylinositol anchor of the variant surface glycoprotein from Trypanosoma brucei brucei.

The glycosylphosphatidylinositol anchor (GPI) from the membrane form variant surface glycoprotein (mfVSG) of Trypanosoma brucei brucei was isolated and identified after radioactive labeling with [3H]myristic acid, by immunostaining on HPTLC with a polyclonal antibody directed against mfVSG and by negative ion laser desorption and fast atom bombardment mass spectrometry of the GPI anchor before and after peracetylation. For the production of monoclonal antibodies the purified GPI molecule was incorporated into liposomes and injected intrasplenically in BALB/c mice. After fusion with the myeloma cell line X63-Ag 8.653 hybridoma cells were cloned by single cell cloning. The secreted antibodies were characterized by ELISA, Ouchterlony immunodiffusion, and Western blot and used in first immunofluorescent studies.     

Establishing apoptosis resistant cell lines for improving protein productivity of cell culture

The authors established apoptosis resistant COS–1, myeloma, hybridoma, and Friend leukemia cell lines by genetically engineering cells, aiming at more efficient protein production by cell culture. COS–1 cells, which are most widely used for eukariotic gene expression, were transfected with human bcl–2 gene. Both bcl–2 and mock transfected COS–1 cells were cultured at low (0.2%) serum concentration for 9 days. The final viable cell number of the bcl–2 transfected cells was ninefold of that of the mock transfectants. Both bcl–2 and mock transfectants were further transfected with the vector pcDNA- containing SV40 ori and immunoglobulin gene for transiently expressing protein. The bcl–2 expressing COS–1 cells produced more protein than the mock transfected COS–1 cells after 4 days posttransfection.Mouse myeloma p3-X63-Ag.8.653 cells, which are widely used as the partner for preparing hybridoma, and hybridoma 2E3 cells were transfected with human bcl–2 gene. Both bcl–2 transfected myeloma and hybridoma survived longer than the corresponding original cells in batch culture. The bcl–2 transfected 2E3 cells survived 2 to 4 four days longer in culture, producing 1.5- to 4-fold amount of antibody in comparison with the mock transfectants.Coexpression of bag–1 with bcl–2 improved survival of hybridoma 2E3 cells more than bcl–2 expression alone. The bag–1 and bcl–2 coexpressing cells produced more IgG than the the cells expressing bcl–2 alone.Apoptosis of Friend murine erythroleukemia(F-MEL) cells was suppressed with antisense c-jun expression. The antisense c-jun expressing cells survived 16 days at non-growth state.     

Elevated glutamate dehydrogenase flux in glucose-deprived hybridoma and myeloma cells: evidence from 1H/15N NMR

The glutamine metabolism was studied in glucose-starved and glucose-sufficient hybridoma and Sp2/0-Ag14 myeloma cells. Glucose starvation was attained by cultivating the hybridoma cells with fructose instead of glucose, and the myeloma cells with a low initial glucose concentration which was rapidly exhausted. Glutamine used in the experiments was labeled with 15N, either in the amine or in the amide position. The fate of the label was monitored by 1H/15N NMR analysis of released 15NH+4 and 15N-alanine. Thus, NH+4 formed via glutaminase (GLNase) could be distinguished from NH+4 formed via glutamate dehydrogenase (GDH). In the glucose-sufficient cells a small but measurable amount of 15NH+4 released by GDH could be detected in both cell lines (0.75 and 0.31 micromole/10(6) cells for hybridoma and myeloma cells, respectively). The uptake of glutamine and the total production of NH+4 was significantly increased in both fructose-grown hybridoma and glucose-starved myeloma cells, as compared to the glucose-sufficient cells. The increased NH+4 production was due to an increased throughput via GLNase (1.6 -1.9-fold in the hybridoma, and 2.7-fold in the myeloma cell line) and an even further increased metabolism via GDH (4.8-7.9-fold in the hybridoma cells, and 3.1-fold in the myeloma cells). The data indicate that both GLNase and GDH are down-regulated when glucose is in excess, but up-regulated in glucose-starved cells. It was calculated that the maximum potential ATP production from glutamine could increase by 35-40 % in the fructose-grown hybridoma cells, mainly due to the increased metabolism via GDH.     

Oxidative stress is not required for the induction of apoptosis upon glutamine starvation of Sp2/0-Ag14 hybridoma cells

L-glutamine (Gln) withdrawal rapidly triggers apoptosis in the murine hybridoma cell line Sp2/0-Ag14 (Sp2/0). In this report, we examined the possibility that Gln deprivation of Sp2/0 cells triggers an oxidative stress which would contribute to the activation of apoptotic pathways. Gln withdrawal triggered an oxidative stress in Sp2/0 cells, as indicated by an increased accumulation of reactive oxygen species (ROS) and an increase in the intracellular content in protein carbonyl groups. Gln starvation also caused a decrease in the intracellular levels of glutathione (GSH). However, a decrease in GSH was not sufficient to induce Sp2/0 cell death since reducing GSH levels with DL-buthionine-[S,R]-sulfoximine did not affect cell viability. The antioxidant N-acetyl-L-cysteine (NAC), while effective in inhibiting ROS accumulation and oxidative stress, did not prevent the loss in cell viability or the processing and activation of caspase-3 triggered by Gln starvation. On the other hand, NAC did reduce the formation of apoptotic bodies in dying cells. Altogether these results indicate that in Sp2/0 cells, Gln deprivation leads to the induction of an oxidative stress which, while involved in the formation of apoptotic bodies, is not essential to the activation of the cell death program.     

Rapid induction of the intrinsic apoptotic pathway by L-glutamine starvation

While the amino acid L-glutamine is known to play a role in the survival of several cell types, the underlying molecular mechanisms are still poorly defined. We show in this report that L-glutamine starvation rapidly triggered apoptosis in Sp2/0-Ag14 hybridoma cells. This process involved the activation of both caspases-9 and -3, suggesting that L-glutamine deprivation initiated an intrinsic apoptotic pathway in Sp2/0-Ag14 cells. Supporting this idea, the cytosolic release of the mitochondrial proteins SMAC/DIABLO and cytochrome c (Cyt c) was observed, with an initial limited leakage occurring during the first 30 min of L-glutamine deprivation, followed by a greater release after 60 min. The latter occurred simultaneously with the translocation of the pro-apoptotic protein Bax to the mitochondria. Finally, a decline in XIAP levels and the activation of caspases-3 and -9 were observed. Thus, L-glutamine deprivation of Sp2/0-Ag14 cells rapidly triggers intracellular events, which target the mitochondria, leading to the cytosolic release of apoptogenic factors, the activation of caspases-9 and -3, and the commitment to the death program. This work introduces the Sp2/0Ag14 hybridoma as a unique model for the study of the molecular events underlying the pro-survival function of L-glutamine.     

Utilization of leucine methyl ester for the generation of hybridomas producing monoclonal antibodies specific to tumor-associated antigens

In an attempt to obtain monoclonal antibodies specific to tumor-associated antigens. C3H/He mice were immunized with syngeneic MM2 tumor cells, and the primed spleen cells were fused with P3-X63-Ag8.653 myeloma cells. The outgrowth of hybridomas, however, was extremely low and monoclonal antibodies were not obtained. The reason for the low hybridoma growth was studied. It was found that MM2 cells used as the immunogen, the fusion partner myeloma cells and the resulting hybridomas shared at least one tumor-associated antigen, namely Q5 antigen. Because of this common antigen, cytotoxic cells, presumably cytotoxic T lymphocytes, which were lytic to the hybridomas, were induced during the culture for generation of the hybridomas. Removal of lysosome-rich cells, including cytotoxic T lymphocytes, from the primed spleen cells before the fusion by treatment with leucine methyl ester, a lysosomotropic agent, drastically improved the outgrowth of hybridomas. By this method, seven stable hybridoma clones producing monoclonal antibodies specific to tumor-associated antigens were obtained. Two of the seven clones were found to secrete monoclonal IgM species, which reacted with the extra-cellular region of the Q5 antigen. This procedure will be an option when production of monoclonal antibodies specific to cell-surface antigens is intended and outgrowth of hybridomas is unexpectedly low.     

Karyotypic characteristics of a number of clonal lines of an interspecific mouse-mink hybridoma

Using G-banding method, a study was made of the karyotypes of mouse myeloma cell line P3-X63-Ag8.653 and some cell hybrid lines originated from a fusion of mink immunized spleen cells and this myeloma. Normal chromosomes 6, 12 and X were not detected in either examined cell of the parental myeloma. The mink chromosomes are preferentially lost from cells of the hybrid lines. These lines varied significantly from cell to cell and from each other in the retention of some mink chromosomes. The karyological study of the hybrid lines revealed some cytogenetical specificities of mouse chromosome composition that were general for cells of hybrid lines, but atypical for myeloma parental cells. This data suggest the cells of myeloma parental line to take part in the processes of somatic hybridization and/or selection for mink immunoglobulin production nonrandomly.     

番木瓜环斑病毒单克隆抗体的制备及在病毒分型上的初步应用

本试验是用番木瓜环斑病毒(Papaya ringspot virus, PRV)提纯制剂免疫的BALB/c小白鼠脾细胞与Sp~2/o-Ag14骨髓瘤细胞融合,获得三个能稳定传代并分泌抗番木瓜环斑病毒的单克隆抗体的杂交瘤细胞系。其中23H1 McAb的效价较高,用ELISA检测,腹水抗体效价高达1:76800,能被PRV兔抗血清所阻断。这3个杂交瘤细胞系产生的单抗与TMV和CMV无血清交叉反应。它们可把PRV四个毒株初步区分为三个血清型。     

The cultivation of mouse and human lymphoid cells on serum-free media]

The optimum composition of several serum-free media has been established for a long-term cultivation of hybridomas, lymphoid and erythroleukemic cells. The medium DME/F12 appeared to be the medium of choice. It is necessary to supplement the basic medium with lipid and iron transport proteins (bovine serum albumin, transferrin) and peptide hormone (insulin) for obtaining stable results. However, there are differences in successful growth of examined cell lines under serum-free conditions: some of them acquire saturation density comparable with that of the control medium (hybridomas derived from myeloma Sp2/0-Ag14, cell lines K-562, Raji) but other lines do not (hybridoma derived from myeloma NS0/1, cell lines Namalwa, RPMI 1788, Molt-4). Thus, these serum-free media are not universal, therefore each new hybridoma and cell line should be tested to determine the suitability for them of some proposed media. The high effectiveness of cultivation under serum-free conditions can be presumably achieved by optimization of both qualitative and quantitative composition of the serum replacement and of the basic medium.     

Monoclonal antibodies against amyloid fibril protein AA. Production, specificity, and use for immunohistochemical localization and classification of AA-type amyloidosis

Monoclonal antibodies against amyloid fibril protein AA were produced by cell fusion of murine P3 X 63-Ag8.653 myeloma cells with spleen cells of immunized Balb/c mice. To increase immunogenicity, protein AA was coupled to horseradish peroxidase (HRP) or human high molecular weight kininogen (HMWK). Using micro-ELISA (enzyme-linked immunosorbent essay) seven hybridoma cell lines secreting antibodies that specifically bind to protein AA have been selected and cloned. When applied to formalin-fixed paraffin sections of a variety of different amyloid types using immunoperoxidase methods, five monoclonal antibodies bound specifically and strongly to amyloid only of the AA type. Since a series of different AA-amyloids could be stained, these reagents may be used to routinely diagnose AA-amyloidosis in tissue sections. A monoclonal antibody against HRP has also been produced that has been utilized to develop a monoclonal peroxidase-antiperoxidases (PAP) complex. When three immunoperoxidase methods were compared, the sensitivity of a conventional rat PAP was comparable to the monoclonal PAP complex, but the latter was easier to handle. Both methods were more sensitive than the indirect immunoperoxidase technique.     

Colcemid treatment of myeloma prior to cell fusion increases the yield of hybridomas between myeloma and splenocyte

Effect of Colcemid treatment of myeloma (X63-Ag8-6.5.3.) prior to fusion with mouse spleen cell was studied in terms of hybridoma formation. Spleen cells from BALB/c mice immunized with various soluble antigens were fused with the myeloma cells by using polyethylene glycol solution. Colcemid treatment of myeloma cells prior to fusion increased the average number of hybridoma colonies per well by 26-570%. The yield of hybridomas producing antigen-specific antibodies was also higher with the Colcemid treatment. The results suggest that most of the proliferative hybridomas are formed by fusion of cells in the M-phase of the cell cycle.